Elucidating Changes in Surface Marker Expression of Dendritic Cells Following Chemical Allergen Treatment Ben C. Hulette, Cindy A. Ryan, and G. Frank Gerberick 1 The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253-8707 Received February 1, 2002; accepted April 22, 2002 Elucidating Changes in Surface Marker Expression of Dendritic Cells Following Chemical Allergen Treatment. Hulette, B. C., Ryan, C. A., and Gerberick, G. F. (2002). Toxicol. Appl. Pharma- col. 182, 226 –233. Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in lymphoid and many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC play a critical role in the induction of contact der- matitis and therefore have become a focal point for the develop- ment of in vitro cell-based methods for contact sensitization test- ing. Because of the low abundance of skin-derived LC, methods to culture DC from peripheral blood are being used by investigators to generate LC surrogates to examine the effects of sensitizing chemicals on APC. It has been reported recently that chemical allergens can induce changes in the expression of various DC surface markers and it has been suggested that the measure of these changes in surface marker expression following allergen treatment could provide the basis for an in vitro test method to predict the contact sensitization potential of a chemical. For the work presented here, DC were differentiated from human periph- eral blood mononuclear cells (PBMC-DC) in culture medium containing GM–CSF and interleukin (IL)-4 to ensure an immature phenotype or were derived from the KG-1 cell line (KG-1 DC) using a defined cytokine cocktail consisting of GM–CSF, IL-4, Flt-3/Flk-2-ligand, thrombopoietin, stem cell factor, and tumor necrosis factor- (TNF). Surface marker expression (HLA-DR, CD54, CD80, and CD86) on these DC was measured by flow cytometry after 48 h treatment with the known chemical allergens dinitrofluorobenzene (DNFB) and methylchloroisothiazolinone/ methylisothiazolinone (MCI/MI), the irritant sodium dodecyl sul- fate, lipopolysaccharide (LPS), and TNF. Treatment of PBMC-DC with either MCI/MI or DNFB induced a slight upregu- lation of class II major histocompatibility (MHC) expression (HLA-DR), whereas LPS and TNF significantly upregulated CD54 and slightly upregulated CD80 and HLA-DR expression. For KG-1 DC, only MCI/MI upregulated CD86 expression, whereas TNF upregulated CD54 and slightly upregulated CD80 and CD86 expression. SDS had no effect on surface marker expression in either PBMC-DC or KG-1 DC. Changes in surface marker expression in PBMC-DC treated with chemical allergens were detected in two of five donors, suggesting a limited sensitivity of PBMC-DC under these defined isolation and culture conditions. Furthermore, we found that the presence of GM-CSF and IL-4 during chemical allergen treatment masked the ability to detect changes in surface marker expression. Our data suggest that, under these culture and treatment conditions, measurement of surface marker changes in vitro using PBMC-DC or KG-1 DC does not provide a sensitive in vitro method with sufficient dy- namic range for assessing the contact sensitization potential of a chemical. © 2002 Elsevier Science (USA) Key Words: contact sensitization; in vitro alternatives; dendritic cells. Dendritic cells (DC) 2 are a distinct group of potent antigen- presenting cells that are bone marrow derived and found in the circulation and within lymphoid and nonlymphoid tissues (Steinman, 1991; MacPherson, 1989). DC have been closely associated with the initiation of primary immune responses, graft rejection, autoimmune diseases, and generation of T-cell- dependent humoral immune responses (Steinman, 1991). In vivo, DC undergo a maturation process that includes pheno- typic and functional changes such as increased cell surface expression of major histocompatibility complex (MHC), co- stimulatory, and adhesion molecules (Romani et al., 1989), which correlates with their ability to activate naive T cells. Verhasselt et al. (1997) showed that lipopolysaccharide (LPS) from gram-negative bacteria upregulated costimulatory molecule expression and, more recently, investigators have shown that the proinflammatory cytokine tumor necrosis fac- tor- (TNF) could alter phenotypic marker expression and induce DC maturation in vitro (Aiba et al., 1997; Coutant et al., 1999; Manome et al., 1999; Rougier et al., 2000). Phenotypic changes in MHC, costimulatory, and adhesion molecules have been observed in cultured DC after exposure to chemical 1 To whom correspondence should be addressed at The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, Ohio 45253-8707. Fax: (513) 627-0400; E-mail: gerberick.gf@pg.com. 2 Abbreviations used: DC, dendritic cell; DNFB, dinitrofluorobenzene; GM–CSF, granulocyte macrophage– colony stimulating factor; IL, interleukin; LPS, lipopolysaccharide; MCI/MI, methylchloroisothiazolinone/methyli- sothiazolinone; MHC, major histocompatibility complex; PBMC-DC, DC differentiated from human peripheral blood mononuclear cells; PGE 2 , prosta- glandin E 2 ; TGF1, transforming growth factor-1; TNF, tumor necrosis factor-. Toxicology and Applied Pharmacology 182, 226 –233 (2002) doi:10.1006/taap.2002.9447 226 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.